Substantial personal and financial costs are associated with congenital and acquired loss of oral tissue structure and function, yet few effective therapeutic strategies for the repair or regeneration of oral tissues are available. Autologous tissue transplantation is severely limited by the availability of sufficient donor tissue and donor site morbidity. The effectiveness of allogenic transplant materials is limited. Tissue engineering combines principles of chemistry, biology and engineering to effect solutions to these problems. Strategies include direct local delivery of tissue inducing signaling molecules, genes for a variety of key proteins and cell/carrier constructs. However therapy based upon delivery of tissue inductive signaling molecules such as the bone morphogenetic proteins or genes (gene therapy) is still in development and the clinical indications for these approaches will be limited. Therefore methods to produce substitute tissue transplants are being developed and we have begun to apply these strategies and methods to the restoration of oral tissue structure and function. Our immediate goal is to develop cell-based tissue engineering for the repair or regeneration of specific oral tissues; dental pulp, dentin and bone. The ultimate clinical utility of a cell based approach to tissue engineering depends in part upon the availability and accessibility of cell capable of appropriate differentiation and morphogenesis. Therefore this project asks the question; do cells need to be derived from the tissue targeted for repair or can cells derived from easily biopsied tissues be utilized? Little is known about the capacity of cells cultured from oral tissues such as pulp and gingiva to effect tissue repair in vivo, however recent data reveal that the potential of cells varies with tissue source. Our general hypothesis is that there are optimum cell/carrier constructs (neotissues) for the development of specific, mature adult tissues in vivo. To test this hypothesis in the context of developing neotissues for the regeneration of dental pulp, dentin, and bone, our specific aims are: 1) optimize basal in vitro development parameters and characterize the tissue formed in vivo by human, gingival, dental pulp and marrow stromal neotissues using two prototype scaffolds, 2) study the expression of osteoblast and odontoblast differentiation genes, and 3) study the effects of signaling molecules associated with bone and dentin regeneration in vivo on oral neotissue development in vitro and in vivo. Data from these experiments is expected to lead to the design of clinical trials testing the safety and efficacy or oral neotissues as well as provide essential data for the use of neotissues as in vitro and in vivo models of reparative tissue formation.